Despite efforts to develop novel treatment strategies, refractory and relapsing sarcoma, and high-risk neuroblastoma continue to have poor prognoses and limited overall survival. Monocyte-derived dendritic cell (DC)-based anti-cancer immunotherapy represents a promising treatment modality in these neoplasias. A DC-based anti-cancer vaccine was evaluated for safety in an academic phase-I/II clinical trial for children, adolescents, and young adults with progressive, recurrent, or primarily metastatic high-risk tumors, mainly sarcomas and neuroblastomas. The DC vaccine was loaded with self-tumor antigens obtained from patient tumor tissue. DC vaccine quality was assessed in terms of DC yield, viability, immunophenotype, production of IL-12 and IL-10, and stimulation of allogenic donor T-cells and autologous T-cells in allo-MLR and auto-MLR, respectively. Here, we show that the outcome of the manufacture of DC-based vaccine is highly variable in terms of both DC yield and DC immunostimulatory properties. In 30% of cases, manufacturing resulted in a product that failed to meet medicinal product specifications and therefore was not released for administration to a patient. Focusing on the isolation of monocytes and the pharmacotherapy preceding monocyte harvest, we show that isolation of monocytes by elutriation is not superior to adherence on plastic in terms of DC yield, viability, or immunostimulatory capacity. Trial patients having undergone monocyte-interfering pharmacotherapy prior to monocyte harvest was associated with an impaired DC-based immunotherapy product outcome. Certain combinations of anti-cancer treatment resulted in a similar pattern of inadequate DC parameters, namely, a combination of temozolomide with irinotecan was associated with DCs showing poor maturation and decreased immunostimulatory features, and a combination of pazopanib, topotecan, and MTD-based cyclophosphamide was associated with poor monocyte differentiation and decreased DC immunostimulatory parameters. Searching for a surrogate marker predicting an adverse outcome of DC manufacture in the peripheral blood complete blood count prior to monocyte harvest, we observed an association between an increased number of immature granulocytes in peripheral blood and decreased potency of the DC-based product as quantified by allo-MLR. We conclude that the DC-manufacturing yield and the immunostimulatory quality of anti-cancer DC-based vaccines generated from the monocytes of patients were not influenced by the monocyte isolation modality but were detrimentally affected by the specific combination of anti-cancer agents used prior to monocyte harvest. Several progressive and relapsing malignancies in pediatric patients have dismal life prognosis. Refractory neuroblastoma and refractory or metastatic sarcoma have an especially poor prognosis, with no consistently curative treatments available. Oberlin et al.  publisheex vivo manipulated dendritic cells producing interleukin-12 in children, adolescents, and young adults with progressive, recurrent, or primarily metastatic high-risk tumors” (EudraCT number 2014-003388-39). The primary endpoint of the trial was an assessment of safety by analysis of the frequency of occurrence of AESI . Vaccines that meet quality control (QC) requirements are registered for use and applied intradermally every 2–4 weeks for up to 35 doses.DCs are essential antigen-presenting cells for the initiation, maintenance, and regulation of immune response . Active 7-108 DCs. Monocytes arise from hematological precursors in bone marrow, with a maturation time of 50–60 h  cytotoxic and targeted anti-cancer therapy preceding monocyte harvest and (ii) variability in the complete blood count on the quality of DC-based anti-cancer immunotherapy in high-risk sarcoma and neuroblastoma patients, representing the two main diagnoses in the DC clinical trial. A secondary aim was to reveal whether monocyte isolation by elutriation is superior to the isolation of monocytes through their adherence to plastic cultivation flasks.9/L, thrombocytes ≥ 75 × 109/L, hemoglobin 80 g/L, estimated glomerular filtration rate (eGFR) ≥ 70 mL/min/1.73 m2, serum creatinine ≤ 1.5-fold the upper limit for the appropriate age, bilirubin ≤ 1.5-fold the upper limit for the appropriate age, AST and ALT ≤ 2.5-fold the upper limit for the appropriate age, ejection fraction ≥ 50%, and fractional shortening ≥ 27% as assessed by echocardiography. In the case of bone marrow infiltration, the allowable ANC was ≥ 0.5 × 109/L and blood platelets 40 × 109/L. In case of liver metastases, AST and ALT had to be ≤ 5-fold the upper limit for the appropriate age. The exclusion criteria were as follows: seropositivity to HIV1,2, Treponema pallidum, hepatitis B or C, known hypersensitivity to the study medication, autoimmune disease that was not adequately treated, uncontrolled psychiatric disease, or uncontrolled hypertension defined as systolic and diastolic blood pressure over the 95th percentile for the appropriate age and height (patients ≤ 17 years old) or ≥ 160/90 mmHg or diastolic blood pressure ≥ 90 mmHg (patients ≥ 17 years old). Patients previously treated with dendritic cells or participating in another clinical trial during the 30 days before enrollment were not eligible to enter this clinical trial.Patient eligibility/inclusion criteria for the clinical trial included being 1–25 years old male/female with histologically confirmed refractory, relapsing, or primarily metastatic high-risk tumors and having a performance status (Karnofsky or Lansky score) ≥ 50 and a life expectancy of longer than 10 weeks. Patients had to be clinically eligible for the surgical procedure to harvest tumor tissue for histological verification and tumor antigen extraction. Female patients had to have had a negative pregnancy test. All patients had to have adequate bone marrow, kidney, liver, and heart function, defined as absolute neutrophil count (ANC) ≥ 0.75 × 10The allowed medication prior to monocyte harvest (leukapheresis) was as follows: metronomic chemotherapy, immune checkpoint inhibitors, and anti-CD20 antibodies were allowed as concomitant medication for any time before leukapheresis. Monoclonal antibodies (except anti-CD20), high-dose chemotherapy, and high-dose corticoids had to have been withdrawn at least 3 weeks prior to leukapheresis with the exception of corticoid treatment of brain edema, which was allowed. Since November 2017, an amendment has been made to the procedure for monocyte harvest, and tyrosine kinase inhibitors have to be withdrawn according to their half-life: drugs with a short half-life of 3–14 h must be withdrawn at least 2 days before leukapheresis , drugs with a medium half-life of 15–35 h at least 7 days before leukapheresis , and drugs with a long half-life of 36–60 h at least 12 days before leukapheresis . Myelopoietic growth factors have to be withdrawn at least 7 days before leukapheresis/monocyte harvest.A precise analysis was performed of preceding and/or concomitant therapy 60 days before monocyte harvest for clinical trial subjects with neuroblastoma and sarcoma diagnoses. Data were mined from the clinical trial electronic case report form and the subjects' medical records. We particularly focused on therapeutic agents with a potential impact on the generation of DCs from monocytes and on DC immunostimulatory properties. These agents and the reports on their role in monocyte biology are summarized in Dendritic cell vaccine manufacture encompassed two phases—(i) preparation of tumor lysate as a source of the patient's tumor antigens and (ii) preparation of monocyte-derived DCs and their loading with tumor lysate. Quality control tests evaluated safety (negativity for pathogens), identity (cell immunophenotype), viability, and functions . The flow and decision tree of the manufacturing process is shown in Tumor lysate was prepared from the tumor tissue obtained from the patient during curative surgery or extended biopsy. In Clean Rooms, necrotic areas and connective tissue were removed from the tumor tissue with a surgical scalpel, keeping the specimen immersed in buffered solution. The remaining tissue was sliced into fragments of about 0.5 mm with a scalpel and forceps and then further crushed with the back of a syringe. Each suspension of tumor fragments and cells in HBSS was lysed through repeated (5 times) freezing in liquid nitrogen and thawing at 37°C. The crude tumor lysate was centrifuged at 450 g/7 min/4°C to remove particulate components. The tumor lysate was released for DC manufacture if the following criteria were met: (i) presence of viable tumor cells reported by a histopathologist, (ii) protein concentration, and (iii) microbiological sterility.9/L before the initiation of leukapheresis. To prevent risk of bleeding or ischemic complications during and after the procedure, hemoglobin of at least 80 g/L and platelets of at least 30 × 109/L were required. In case of a patient with a body weight of < 20 kg, the leukapheresis set was pre-filled with donor erythrocytes. The aim of the leukapheresis was to obtain 60–80 mL of concentrate of mononuclear cells with a content of at least 0.5 × 109 monocytes. Subsequent addition of 5% human albumin to the minimum required volume of 80 mL for further processing was allowed.Monocytes were harvested as part of the mononuclear white blood cell (WBC) fraction. Mononuclear cells were collected from the peripheral blood of the patient using the Terumo BCT Spectra Optia Apheresis System. For collection, we used either an intermittent or continuous leukapheresis system. Due to its superior collection efficacy and easier procedure settings, we have preferred the continuous leukapheresis system since April 2018. A citrate dextrose solution, solution A (ACD-A), was used as an anticoagulant. In patients with a body weight of < 20 kg, anticoagulation with heparin was used to prevent citrate toxicity. The requirement for the minimal WBC count was 3 × 102 tissue culture flasks with 35 mL of CellGenix® GMP DC Medium at 37°C/5% CO2 and were then washed with HBSS and processed further. Monocytes seeded from the elutriation product or attached by plastic adherence were then cultivated in three 175-cm2 tissue culture flasks with 70 mL of CellGenix® GMP DC medium supplemented with GM-CSF  and IL-4  at 37°C/5% CO2/6 days. On day 3, a fresh 70 mL of medium supplemented with the same concentration of GM-CSF and IL-4 was added to the culture. On day 6, immature DCs were exposed to autologous tumor lysate antigens (10 μg/mL) with added keyhole limpet haemocyanin , IL-4 (320 U/mL), and GM-CSF (1000 U/mL) at 37°C/5% CO2/for 1.5–2 h. Maturation was induced by lipopolysaccharide (200 U/mL) and interferon-γ (50 ng/mL) for an additional 6 h at 37°C/5% CO2. Finally, cells were collected using accutase , counted in a Bürker cell chamber and frozen in aliquots of 2 × 106 DCs in 100 μL of freezing medium CryoStor® CS2 at -80°C. All doses of the DC-based investigational medical product (IMP) named “MyDendrix®” were stored at -150°C until administration to the patient.The numbers of WBCs, B-cells and T-cells, monocytes, and granulocytes in the leukapheretic product were evaluated using a hematology analyzer  and flow cytometer  with staining for CD3  and CD19 . Monocytes for DC manufacture were separated from the leukapheresis product by either elutriation or adherence to a plastic surface. During elutriation , blood cells were separated on the basis of sedimentation velocity into six fractions, where the last fraction rich in monocytes was used for DC manufacture. Contaminating cells after elutriation were mainly granulocytes with similar sedimentation velocity to monocytes. Five hundred million monocytes adhered for 2–4 h in three 175-cm5 cells) were seeded into 1 well of a 6-well culture plate for sensitive adherent cells  and cultured in 3 mL of DC medium for 2 days (37°C/5% CO2) to obtain (i) medium containing cytokines produced by DCs during cultivation and (ii) mature DCs for phenotypic evaluation after 2 days of post-thaw cultivation. A 0.5 mL volume of medium containing DC-produced cytokines was collected after 23–25 h upon DC seeding and was centrifuged (10 min/410 g/4°C), and the supernatant was stored at -25°C for no longer than 30 days prior to analysis. For immunophenotypic evaluation of mature DCs, both detached and adherent DCs were harvested 47–49 h after DC seeding. The culture medium was collected and pooled with DCs harvested by accutase (0.5 mL/well 8.87 cm2/37°C) and centrifuged (5 min/410 g/20°C). The pellet was resuspended in 800 μL HBSS with 0.25% human albumin (Grifols) and processed immediately for immunophenotypic evaluation. Viability quantification was performed by propidium iodide (PI) exclusion assay. Briefly, 105 DCs were stained with 10 μL of 1% PI in HBSS followed by immediate flow cytometric (Cytomics FC500) analysis of PI-positive events (= non-viable cells). The immunophenotype of DCs was evaluated in post-thaw DCs and in post-cultivation mature DCs. For the detection of each surface molecule, 0.5 × 105 DCs were incubated for 20 min in the dark with the following antibodies: CD80-PC7 , CD83-FITC , CD86-PE , CD197-PE , HLA-DR-PC5 , CD14-PE , or isotype controls IgG-PC5 , IgG-PC7 , IgG2a-FITC , or IgG2a-PE , all from Beckman Coulter. Flow cytometric analysis was performed using a Cytomics FC500 with CXP software by manual gating on individual parameters, and the discrimination by appropriate isotype control was used to gate and quantify positive events. The concentrations of IL-12 and IL-10 in the DC culture medium were measured by flow cytometric bead assay (BD Biosciences) using internal quality controls . Absolute production of IL-12 or IL-10 per 106 DC and the IL-12/IL-10 ratio were calculated. The allogenic  and autologous (auto) stimulatory properties of DCs were examined by mixed lymphocyte reaction (MLR). In allo-MLR, the target cells were the peripheral blood mononuclear cells (PBMCs) obtained from pooled buffy coats from healthy donors. In auto-MLR, the target cells were the patient's lymphocytes separated by centrifugation in a density gradient using Histopaque-1077  from the leukapheresis product obtained for DC manufacture. These pre-vaccination lymphocytes were cryopreserved using CryoStor CS5 medium (BioLife solutions) at -150°C and thawed prior to auto-MLR seeding. A sample of 107 target lymphocytes were stained with 250 μL 10 μM carboxyfluorescein succidimidyl ester  and seeded into a sterile 96-well culture plate  at 105 cells/well in 200 μL of complete X-vivo 10 medium (Lonza) containing 5% inactivated human male AB serum (SigmaAldrich) for the following: (i) 104 DC/well in 10:1 target:effector MLR, (ii) positive control (PC) with phytohemagglutinin  at a final concentration of 10 μg/mL, or (iii) negative control (NC) with complete X-vivo medium only. MLR experiments were seeded in triplicate and cultured for 6 days at 37°C/5% CO2. 2 × 104 cells from each well were stained with CD3-PC7  for flow cytometric detection of CFSE fluorescence on CD3+ T cells. Discrimination for dividing cells was set up using NC. T-cell proliferation was calculated as follows: [(average % of dividing T-cells in 10:1 MLR) – (average % of dividing T-cells in NC)] × 100/[(average % of dividing T-cells in PC) – (average % of dividing T-cells in NC)].DC characteristics were evaluated as a part of the quality control process of IMP from an aliquot of manufactured DC from each batch. The cryotube with DC was removed from a deep freezing box (-150°C) into a laminar flow box, quickly and gently thawed in hand while avoiding shaking, 1 mL of cold (2–8°C) DC medium (CellGenix® GMP-grade) was slowly added to the thawed DCs, and the DC suspension was transferred into 2 mL of cold DC medium. The DC suspension was handled at room temperature and processed immediately. DCs . software .As of May 2019, 47 subjects were enrolled in the clinical trial, and the manufacturing process of DC-based vaccine was performed in 31 cases. Of these 31, the most common diagnoses were sarcoma, with 19 cases (61%), and high-risk neuroblastoma, with 4 cases . In thisMycoplasma spp. negativity, the viability and phenotype of thawed DCs, the phenotype of thawed DCs after 2-day cultivation, the production of IL-12 and IL-10 during 24-h cultivation of thawed DCs, and 6-day allo-MLR and auto-MLR. All batches of DCs fulfilled the microbiological criteria of QC and the criteria of viability, ranging from 85 to 100% with a mean of 95%. Their variability in phenotype and immunostimulatory property is shown in 6 DC , IL-10 280 pg/106 DC , and IL-12/IL-10 ratio 35 (1–246). The mean in vitro proliferation of T-cells stimulated by manufactured DCs was 67% (29–98%) in allo-MLR and 9% (−3–37%) in auto-MLR. Due to inappropriate results for the immunostimulatory parameters of QC , six out of 22 (27%) of the manufactured batches of DCs were not released for use in the clinical trial. The parameter values of the manufactured batches of DCs are shown in We achieved DC yields ranging from 0 to 43.6%, with a mean of 17.2% and an s.d. of 12.7% in this specific cohort. A DC yield equal to 0 represented a manufacturing process that was unsuccessful, with all DCs detached from the flasks. The quality control parameters involved microbial sterility and Isolation of monocytes for DC manufacture was performed by elutriation in 14 cases and by plastic adherence in nine (39%) cases based on the real-world situation. Until March 2017, we performed elutriation of the leukapheresis product in all cases . Between April and September 2018, we performed elutriation in cases KDO-0121, -0137, and -0139, and adherence to plastic in cases KDO-0133, -0142, and -0144 due to there being > 10% neutrophils in the leukapheresis product or technical issues with the Elutra device for KDO-0119 and -0131. After October 2018, we isolated monocytes exclusively by adherence to the plastic surface in all cases: KDO-0135, -0141, -0144, and -0147.p = 0.643). Thus, we conclude that the isolation of monocytes by adherence to plastic is comparable to a manufacturing process with monocyte elutriation.Addressing the issue of whether the elutriation process is superior to adherence to plastic retrospectively, we compared the proportions of batches passing QC and their DC yield and phenotypic and immunostimulatory properties under the two methods. Adherence to plastic resulted in two (22%) batches not being released, and elutriation resulted in five (36%) batches not being released (four did not pass QC and one was not manufactured). The OR (odds ratio) for passing QC in the plastic-adherence modality was 1.94 (95% CI: 0.29–13.19). The DC yield, viability, phenotype, and immunostimulatory properties  in adherence to plastic vs. elutriation are summarized in p = 0.046). DC yield was not associated with any single parameter of CBC. Expression of CD14 on manufactured cells was negatively correlated with relative lymphocyte count in CBC (p = 0.001) (p = 0.010) and NRBC (p = 0.018) in pre-leukapheresis CBC (p = 0.016) (p < 0.001) and low proportion of T-cells (p = 0.001) in the leukapheresis product were associated with increased expression of CD14 on manufactured cells (p = 0.027) (With the aim of identifying the CBC parameters (shown for each batch in = 0.001) . The levesis CBC . The lev= 0.016) . Similared cells . A high = 0.027) .n = 17) for administration of therapy potentially interfering with monocyte viability and/or differentiation, namely TKI, mTOR inhibitors, chemotherapy in cell biology-interfering doses, i.e., MTD-based dose, anti-RANKL mAb, retinoic acid, and/or G-CSF (n = 6) for metronomic therapy/chemotherapy or no potentially monocyte-interfering therapy concomitantly or < 60 days prior to monocyte harvest. All batches from the “0” category passed QC, whereas seven out of 17 (41%) monocyte-derived DCs from the “m” category failed to be released for patient administration. The OR for passing QC in category “0” was 9.3 (95% CI: 0.5–191). DC yield, DC immunophenotype on day 0 and day 2, and production of IL-10 did not differ between the “0” and “m” categories (6 DCs in the “0” category and 743 pg/106 DCs in category “m” (p = 0.083). The median IL-12/IL-10 ratio was 71 in the “0” category and 9 in the “m” category (p = 0.002). The median T-cell proliferation in allo-MLR was 86% in the “0” category and 63% in the “m” category (p = 0.027), and the in auto-MLR was 12% in the category “0” and 5% in category “m” (p = 0.036)  .In the analyzed study cohort, therapeutic regimens were heterogenic, with patients often treated with a combination of various compounds prior to monocyte harvest, and thus further categorization into single agent-defined subgroups and their analysis were impossible. Therefore, we performed cluster analysis of DC parameters in the context of therapy prior to monocyte harvest . Here weHere we show that despite strict adherence to the validated manufacturing protocol, the outcome of the manufacture of the medicinal product with monocyte-derived DCs is highly variable in terms of both DC yield and immunostimulatory properties. Moreover, in 30% of cases, manufacture of DC-based immunotherapy for advanced sarcoma and high-risk neuroblastoma patients resulted in a product that did not meet the specifications for the medicinal product and therefore was not released for application. This product failure rate was higher than in published studies , 24. ThuRegarding the method of monocyte isolation, we assessed whether monocyte extraction by a simple method of adherence to a plastic surface is comparable to the elaborate method of elutriation. During elutriation, monocytes can be contaminated with granulocytes with a similar sedimentation velocity to monocytes. Based on this observation, we validated the DC-manufacturing process with isolation of monocytes by adherence to plastic  to avoidin vitro impairs the immunostimulatory properties of monocytes, including up-regulation of the immunoinhibitory surface molecule ILT-3 and decreased capability to up-regulate MHC II in response to LPS (in vivo with pazopanib without any other immediate treatment (KDO-0101) did not result in attenuated DC vaccine quality. Topotecan has been shown to partially activate monocyte-derived DCs but to prevent the full maturation of DCs stimulated with a cocktail of proinflammatory mediators  and to be further enhanced through the addition of TMZ  a low relative lymphocyte count, (ii) a high relative neutrophil count in CBC, and (iii) a high proportion of monocytes in the leukapheresis product, were associated with unfavorably high expression of CD14 on the manufactured cell product. Moreover, the presence of an increased number of immature granulocytes was associated with decreased potency of the DC-based product as quantified by allo-MLR. These observations may be underlain by emergency myelopoesis stimulated by G-CSF, which leads to a quantitative and qualitative change in all circulating myeloid cell types including neutrophils, monocytes, and myeloid-derived suppressor cells , 35. WhiHere, we show that treatment of patients with certain anti-cancer agents in MTD-based doses prior to monocyte harvest often leads to failure of manufacture of the immunostimulatory DC-based vaccine. We propose that the optimal time for monocyte harvest for generating DCs is prior to a cell-interfering treatment. With respect to the DC-manufacturing workflow, this would mean, in a majority of cancer patients, the implementation of DC manufacture from cryopreserved monocytes. Several studies have investigated the effect of cryopreservation on monocyte differentiation into DCs, but results have been conflicting. Some studies observed cryopreservation to have no effect on monocyte-derived DC production , 41. On Another issue in the context of the concurrence of anti-cancer treatment and monocyte-derived DC manufacture is the length of the pharmacotherapy-free period prior to monocyte harvest. From our real-life experience gained on this study group, we conclude that a 30-day interval without treatment is not sufficient for the combination of temozolomide and irinotecan to sufficiently wash out the monocyte biology-interfering effect of this combination. However, the issue of a safe therapy-free window is not likely to be addressable through the establishment of a wash-out period for a particular drug. The fitness of monocytes and their capacity to differentiate and mature into DCs with high antigen-presenting effect is a matter of their biological function in the context of iatrogenic affection, which is complexly shaped by the need for immediate treatments, their combinations, their cumulative doses, and the long-term history of treatment. Therefore, identifying a marker revealed from a patient's peripheral blood that predicts the outcome of DC-generation would help to avoid an unproductive anti-cancer DC-manufacturing process. Here we show that a high monocyte count in CBC is not predictive of an efficacious outcome for DC generation. Nevertheless, we find that the presence of immature granulocytes in CBC may predict decreased immunostimulation elicited by DCs and, subsequently, unsuccessful preparation of DC-based IMP. However, closer evaluation of monocyte function prior to their collection for DC generation may be considered. A surrogate marker for the immunostimulatory capacity of monocytes may be evaluated in (i) their phenotype, e.g., the level of HLA-DR or ILT-3 expression on monocytes or the proportion of particular monocyte subsets according to CD14 and CD16 expression, or (ii) their ability to produce pro-inflammatory cytokines upon TLR stimulation .in vitro DC generation via induction of immunogenic cell death and in vivo for effector response of DC-activated T-cells but also with respect to optimal monocyte immunostimulatory functions. Finally, these findings may also have implications for the general pharmacology of anticancer treatment. As our model of ex vivo-activated DC preparation generally parallels the in vivo differentiation pathways of monocytes to the antigen-presenting cells, we may imply that drug combinations at doses used clinically may result in an impairment of patient DCs and possibly immune competence in general. In conclusion, these findings may stimulate further research on dose and mechanism-of-action-based drug combination in patient-centered trials to optimize the treatment modalities currently available in clinical oncology.In summary, monocytes represent a key starting material for anti-cancer DC-based vaccine manufacture. Therefore, monocyte conditions have an impact on the manufacturing yield, the differentiation into DCs, and the level of maturation and subsequent immunostimulatory functions. For DC manufacture from heavily pretreated pediatric patients with high-risk sarcomas and neuroblastoma, we conclude that the manufacturing yield and immunostimulatory quality of anti-cancer DC-based vaccine generated from patient's monocytes were not influenced by the monocyte isolation modality but were detrimentally affected by certain combinations of anti-cancer agents. Thus, the combination of chemotherapy or targeted therapy with DC-based immunotherapy needs to be scheduled not only with respect to the likely beneficial role of anti-cancer agents on the immunogenicity of tumor antigens for both All datasets generated for this study are included in the manuscript/The studies involving human participants were reviewed and approved by Ethics Committee, University Hospital Brno. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.EH contributed to the trial design, contributed to the study design, participated in clinical data acquisition and analysis, contributed to Supplementary Material preparation, and drafted the manuscript. KP supervised IMP manufacture, contributed to laboratory data acquisition and analysis, contributed to data interpretation, and drafted the manuscript. DC participated in clinical data acquisition, contributed to Supplementary Material preparation, and revised the manuscript. IS performed statistical analysis, contributed to figure preparation and data interpretation, and drafted the manuscript. PMu contributed to the trial design, performed patient enrollment and treatment, contributed to data interpretation, and drafted the manuscript. PMa contributed to the trial design, participated in patient treatment, and drafted the manuscript. LFe contributed to laboratory data acquisition and analysis, contributed to Supplementary Material preparation, and drafted the manuscript. JM contributed to the trial design and drafted the manuscript. LJ participated in IMP manufacturing and revised the manuscript. LSe contributed to IMP manufacturing—monocyte harvest and drafted the manuscript. RP contributed to IMP manufacturing—starting material harvest and revised the manuscript. LFl contributed to IMP manufacturing—certification and revised the manuscript. LSo contributed to the trial design and revised the manuscript. RD, JS, and DV contributed to the trial design, contributed to data interpretation, and revised the manuscript. LZ-D conceived the study design, designed and supervised laboratory data acquisition and analysis, contributed to data analysis and interpretation, and drafted and finalized the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Evidence regarding the relationship between synthetic house-tree-person (S-HTP) drawing test and depression in cancer patients is limited. The aim of this study was to explore the applicability and validity of S-HTP drawing test in cancer patients suffering from depression. As a population based cross-sectional study, 167 patients with cancer were enrolled in a hospital in China from December 2015 to December 2017. Self-edited general information questionnaire, self-rating depression scale (SDS), and the S-HTP drawing test were completed by all participants.χ2 = 68.657, P < 0.001. Nagelkerke R2= 0.466). Correlation analysis revealed a positive correlation between S-HTP drawing test and SDS for depression state of cancer patients (p < 0.01). The average age of 167 selected participants was 52.92 ± 10.43 years old, and about 58.7% (98/167) of them were male. On SDS, depression rate was found in 34.1% (27/167) cancer patients. The logistic regression equation was established by using the depression drawing characteristics as the independent variables and the evaluation results from SDS as the dependent variable and 9 drawing characteristics employed in the regression equation ( There are interrater reliability and test-retest reliability between S-HTP drawing test and SDS. The S-HTP drawing test could help in screening depression in cancer patients. The increase in morbidity and mortality of cancer has made it the leading cause of death in China since 2010 , 4. A meIn 1948, Buck  has firsThe cross-sectional study was conducted among 200 participants at the Second Xiangya Hospital of Central South University in Changsha, Hunan Province, China, from December 2015 to December 2017, who agreed to participate in the study. Thirty-three patients were excluded from the study because they failed to complete the self-rating depression scale (SDS) or S-HTP drawing test, and 167 cancer patients were finally included.The inclusion criteria were as follows: (1) age ≥ 16 years old, (2) cancer pathology and under treatment for cancer, (3) having reading comprehension ability, and (4) consent to participate in the study. The exclusion criteria included cancer patients with severe organ dysfunction and poor physical condition who could not complete the test. Patients with a professional basis in drawing and those familiar with the rules of the drawing test were not included. In addition, patients who were under treatment for depression were excluded.All patients underwent detailed face-to-face clinical interviews. Self-edited general condition questionnaire, SDS, and S-HTP drawing test were used in the interview. The participants first did the S-HTP drawing test. After that, these cancer patients were allowed for 5 to 10 minutes to rest. Then, the self-edited general information questionnaire and SDS were fulfilled.7 items including age, gender, nation, education, marital status, occupation, and income consisted of the questionnaire .The SDS  is a 20-In our previous published study, the whole process of S-HTP drawing test was described in detail . All of Based on R.C. Burns' drawing test interpretation system and other relevant literature, we identified a set of drawing features related to depression –19. A toThe intertester reliability and test-retest reliability were used to evaluate the reliability of S-HTP test drawing features in this research. Intertester reliability and test-retest reliability are expressed by the Kappa coefficient and correlation coefficient r, respectively. The value of the Kappa coefficient ranged from 0.752 to 1.000, and r of 20 patients retested was 0.710 to 0.857, which means the results of the drawing features were stable and reliable. In order to represent the S-HTP drawing test validity, the criterion validity was used in this study. The data of depression from cancer patients identified by S-HTP drawing test and SDS scale was used to perform the correlation analysis. The result of SDS scale was regarded as the “gold standard” in this study, and the validity coefficient was regarded as the correlation coefficient.χ2 or Fisher's exact test, when appropriate. The role of S-HTP drawing characteristics in predicting depression was examined with the logistic regression equation. A value of P less than 5% is of statistical significance.The data were analyzed with SPSS version 19 for Windows . Single-sample Kolmogorov-Smirnov test was used to test whether the continuous variables are normally distributed. The mean ± SD (standard deviation) was used to represent the continuous variables which conform to the normal distribution, and the median (interquartile range [IQR]) was used to represent the nonnormal variables. Two independent samples Student's t-test were used to compare the mean of two continuous variables which conform to normal distribution. Mann-Whitney U test and Kruskal-Wallis test were used to compare the mean values of 2 groups, 3 groups or more groups of nonnormal distribution variables. The frequencies of categorical variables were compared using Pearson Initially, a total of 200 patients were enrolled in the study, and 33 cases of them were excluded due to missing questionnaires or drawings. Finally, 167 patients with cancer were included in the study, and the effective rate was 83.5%. Among the 167 cancer patients, 98 patients were male, accounting for 58.7%, and 69 were female, accounting for 41.3%. Their age ranged from 16 to 72 years, with an average age of (52.92 ± 10.43) years. The general characteristics of all patients are shown in The score of SDS ranged from 25.00 to 80.00, with an average of (42.88 ± 10.43) in this study. Compared to the norm standard score  . To explore the relationship between the characteristics of S-HTP drawing test and depression in cancer patients, a total of 9 drawing features were included in the equation. The total test results of the model coefficients revealed that χ2=68.657, P < 0.001, indicating that the established logistic regression equation model was statistically significant. Based on the regression coefficients shown in ∗D1+0.919∗D2+2.044∗D3-0.888∗D5-0.944∗D6+1.439∗D9+2.106∗D10+0.679∗D14+1.148∗D23. The Nagelkerke R2 value was determined to be 0.466, showing good fitness. These results indicate that the equation established in this study have a good level of interpretation, and the S-HTP drawing features were suitable for the assessment of depression in cancer patients.In this study, 11 depressive drawing features  were used as independent variables, and the results of the SDS (setting the depression group to 1 and the depression-free group to 0) were used as the dependent variable, then, a logistic regression model was established (χ2=[(|ad-bc| -n/2)2n]/[(a+b)(c+d)(a+c)(b+d)]=[(|32 × 98-25 × 12| -167/2)2 ×167]/(57×110×44×123)=37.29. χ2>6.63, P<0.01 . At the same time, the logistic regression equation was performed in 110 cancer patients without depression, and the finding demonstrated that 98 cancer patients did not have depression, with a correct rate of 89.1% (98/110). The total correct rate was 77.8% (130/167), p < 0.05), and there were no differences in SDS scores in other subgroup analyses (p > 0.05)  .Depression refers to a painful experience when an individual feels sad, distressed or discouraged. Its characteristic symptoms include the disappearance of pleasure, helplessness, despair, self-guilt and suicidal tendencies . DepressMost human thoughts and mental activities can be visualized. For example, the presence of physical objects can significantly enhance memory. Therefore, mental problems, such as depression can be better identified and resolved by analyzing drawings . S-HTP dThe five items with a high incidence of depression assessment items were D8, D17, D20, D4, and D5, suggesting that low mental motivation, pessimistic about real life and melancholy are prevalent in cancer patients. This study found that the correlation coefficient between the result of S-HTP drawing test and SDS was 0.49, and the consistency hypothesis test showed that S-HTP drawing test was positively correlated with SDS. These results showed that S-HTP drawing test was feasible and effective in evaluating depression in patients with cancer. This can also be proved by the logistic regression equation established to screen depression. The nine painting features included in the regression equation were D1, D2, D3, D5, D6, D9, D10, D14, and D23. The detail explanation and psychological meaning of these items are shown in The occurrence of depression in cancer patients could be attributed to the diagnosis of cancer itself, the heavy economic burden of treatment and poor therapeutic effect. Depression often results in severe consequences if it is not detected early and managed effectively in cancer patients. Therefore, it is necessary to monitor the occurrence of depression in cancer patients and to establish valid evaluation methods. The S-HTP drawing test can effectively reveal the inner emotion of cancer patients because it can be performed with less psychological defense as opposed to using the questionnaire alone. In addition, the S-HTP drawing test is a novel concept which can be an effective way of communication for researchers and patients who are unwilling or unable to express their feelings verbally. The S-HTP drawing test will have more application prospects with the further standardization and improvement of modern technology in the future.There are some limitations in this study. First, the study cannot find causal explanations because of its cross-sectional characteristic. Second, this study used the Zung's self-rating scale to screen for depression in cancer patients with reference to the Chinese National Normative Scores. However, the results of the SDS, drawn from self-rating scales, lack comparability to some extent with the clinical diagnostic criteria for depression disorders found in the International Classification of Diseases 10 (ICD10) and Diagnostic and Statistical Manual of Mental Disorders IV (DSM-IV). Third, the different capacity of the S-HTP drawing test to identify more the nondepressed patients than depressed even if there is an interrater and test-retest reliability between S-HTP drawing test and SDS. The clinician should clearly know the capacity of the test to distinguish patients to be pharmacologically treated or not. In addition, we did not perform cognitive function assessment on the subjects before starting this study. If the patient has cognitive impairment, it may affect the outcome. But we only included the patients who had reading comprehension ability. Finally, it is unclear whether studies on other nationalities will yield similar findings for the reason that these findings are based on Chinese patients.S-HTP drawing test and SDS have interrater reliability and test-retest reliability. Our findings indicate that the S-HTP drawing test could help in screening depression in cancer patients.


Citrullus colocynthis (L.) Schrad  extracts as bio-fuels is reported. The structure, optical, and colloidal properties of the synthesized ZnO-NP samples were studied. Results illustrate that the morphology and particle size of the ZnO samples are different and depend on the bio-fuel. The XRD results revealed that hexagonal wurtzite ZnO-NPs with mean particle size of 27–85 nm were produced by different bio-fuels. The optical band gap was increased from 3.25 to 3.40 eV with the decreasing of particle size. FTIR results showed some differences in the surface structures of the as-synthesized ZnO-NP samples. This led to differences in the zeta potential, hydrodynamic size, and more significantly, antioxidant activity through scavenging of 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) free radicals. In in vitro cytotoxicity studies on 3T3 cells, a dose dependent toxicity with non-toxic effect of concentration below 0.26 mg/mL was shown for ZnO-NP samples. Furthermore, the as-synthesized ZnO-NPs inhibited the growth of medically significant pathogenic gram-positive (Bacillus subtilis and Methicillin-resistant Staphylococcus aurous) and gram-negative (Peseudomonas aeruginosa and Escherichia coli) bacteria. This study provides a simple, green and efficient approach to produce ZnO nanoparticles for various applications.In this paper, a green microwave-assisted combustion approach to synthesize ZnO-NPs using zinc nitrate and  Green synthesis is more eco-friendly with less toxic effects in comparison with chemical processes [C. colocynthis a valuable cucurbit plant, broadly distributed in the tropical areas of the world, has many pharmaceutical and nutraceutical values [C. colocynthis has shown very effective antibacterial, anti-inflammatory, antioxidant and anticancer activities [C. colocynthis are usually attributed to its polyphenolic compounds such as isosaponarin, isovitexin and isoorientin 3-O-methyl ether [C. colocynthis could be extremely suitable for drug delivery, gene delivery and biosensor applications, where there is a direct contact of these nanoparticles with blood. This study attempts to exploit C. colocynthis extract as a bio-fuel for the synthesis of ZnO-NPs for biomedical applications.Zinc oxide nanoparticle as a non-toxic, low-cost, and non-hygroscopic metal oxide is very economical and safe polar inorganic crystalline material which has extensive applications in different areas. Nano-sized ZnO particles are an important metal oxide material which has a wide direct band gap 3.37 eV) and large exciton binding energy 60 meV) at room temperature ,2. The Z.37 eV an0 meV at 2O3 , TiO2 [22O3 [2 , CeO2 [2O3 [2 [2  and ZnO rocesses . The C. l values . The frutivities . The theyl ether ,27. ThesC. colocynths were suitable fuels because they contain various compounds such as Cucurbitacins, glycosides, flavonoids and phenolic acids [Zinc oxide nanoparticles using combustion process can be produced through the combination of zinc nitrates in an aqueous solution with a fuel. In the experiments, fruit, seed and pulp extracts of ic acids ,29 that During the formation of ZnO nanoparticles an exothermic reaction takes place between oxidizing and reducing agent. Nitrate could act as an oxidizer agent and fuels could act as reducing agents for the formation of ZnO nanoparticles ,31,32.2+ ions form a complex compound inside the nanoscopic templates of metabolites through transfer of the π electrons from the carbonyl groups to the transition metal–dative coordinate bonding and finally, the ZnO-NPs formed by thermal decomposition of Zn2+ complex (The possible mechanism for formation of ZnO-NPs is as below: The nitrate group is a stronger oxidizer and can oxidize hydroxyl groups present in biomolecules to carbonyl groups and simultaneously Zn complex .h k l); lattice constants a, b, and c, interplanar angle (the angle φ between the planes (h1k1l1), of spacing d1 and the plane (h2k2l2) of spacing d2), and unit cell volumes (V), are calculated from the lattice geometry [The wurtzite lattice parameters, such as the values of d, the distance between adjacent planes in the Miller indices  consistent with the Kubelka-Munk model shown in The UV-vis spectra of ZnO-NPs bio-synthesized by use of fruit, seed and pulp of In this study, zeta potential and hydrodynamic size were measured by DLS. The zeta potential for colloidal suspensions of ZnO-NPs samples were found at −25.60, −19.5 and −15.3 mV for ZnO-F, ZnO-P and ZnO-S, respectively. From the zeta potential values, it is suggested that the NPs were stable and warped with anionic organic phases and responsible for electrostatic stabilization . FTIR re2/Г2), respectively , P. aeruginosa and E. coli. The highest antimicrobial activity was observed against B.subtilis, P. aeruginosa and E. coli, while a lower activity was found against MRSA. This difference can be related to the structural and chemical composition of the cell membrane of micro-organisms. These findings are consistent with earlier studies that studied the antimicrobial activity of ZnO against B. subtilis, P. aeruginosa [E. coli [In this study, the antimicrobial activity of ZnO-NPs synthesized using new bio-fuels was evaluated. In this analysis, the ZnO-NPs showed antimicrobial activity against a range of different bacteria . The ZnOruginosa  and E. c[E. coli ,44.C. colocynthis were collected from Koohzar district, Khuzestan, Iran in April. The whole fruit were oven-dried at 45 °C for 48 h, and then fruit, seed and pulp separately milled to fine powder. About 5 g from each fine powder was dispersed in 50 mL of distilled water and magnetically stirred for 30 min at 100 °C. The extracts were filtered and centrifuged to eliminate any bulks and kept at −20 °C before use.Mature fruits of 3)2 6H2O was dissolved in 2 mL distilled water in a porcelain crucible and then thoroughly mixed with 5 mL of the extract  under stirring and finally was placed under microwave irradiation at 340 W. After boiling and evaporating, the mixture quickly foamed up, and released gases. The whole process was completed in 8 min and the ZnO-NPs were left as residue  in the range of 200–800 nm. Transmission electron microscope  with an acceleration voltage of 120 kV was applied to study size and morphology of the ZnO-NPs. TEM samples were prepared by depositing a few drops of the sample suspension on a copper grid followed with drying at room temperature. The morphology of samples were further observed using FESEM . The small amounts of powder samples were mounted on a metal stub using carbon tape and then gold-coated using a sputter coater. The particle sizes and the zeta potentials of ZnO-NPs were analyzed by photon correlation spectroscopy and laser Doppler anemometry, respectively, with a Zetasizer, Nano ZS  at room temperature.The crystal structure of ZnO-NPs were analyzed by PXRD  in the 2θ range between (2–80°) at room temperature. FTIR spectra of the samples were recorded by FTIR spectrometer , with a resolution of 4.0 cm50) value, defined as the amount of sample that inhibits 50% of cell growth, was calculated from the concentration–response curves.The in vitro cytotoxicity of ZnO-NPs samples was assessed by the method using 3--2,5-dephenyl-tetrazolium bromide (MTT) assay. Briefly, 3T3 cells were seeded at a density of 2 × 105 cells/mL in 96-well microplates and incubated for 24 h. Subsequently, the cells were treated with the various concentrations of samples in the presence of 10% FBS for 24 h. The samples were suspended separately in a stock solution at 5 μg/mL in a solution of dimethyl sulfoxide (DMSO)/double distilled water. After 24 h of incubation, 20 μL of 5 mg/mL MTT in the PBS buffer was added to each well, and the cells were incubated for another 4 h at 37 °C. The 100 μL of DMSO was added to dissolve the formazan crystal formed by live cells. Optical absorbance was measured at 570 nm. Cell viability was calculated as the percentage of absorbent compared to control. The 50% inhibitory concentration  B29 and Methicillin-resistant Staphylococcus aurous (MRSA)) and gram-negative (Peseudomonas aeruginosa (P. aeruginosa) ATCC 15442 and Escherichia coli (E. coli) E266) pathogens were performed through a disc-diffusion assay. Briefly, the bacteria were grown overnight in nutrient broth. The bacterial inoculum was standardized to 0.5 MF units, meaning that approximately 108 colony-forming units of each bacterium were inoculated onto a plate. Previously prepared samples impregnated discs (6 mm) at the various concentrations were placed aseptically on plates inoculated with bacteria and incubated at 37 °C for 24 h. After incubation, the zone of whole inhibition was measured. All tests were replicated three times.In the present study, in vitro antimicrobial activities of prepared ZnO samples towards gram-positive (C. colocynthis through microwave-assisted combustion. The bio-fuels could effectively mediate the nucleation and growth of ZnO nanoparticles under microwave radiation. Results reveal that the bio-fuels significantly affect the morphology of ZnO. Flower-like, hexagonal, and block-shaped nanostructures were produced by fruit, seed and pulp, respectively. The particle size of nanoparticles was varied between ~27 to ~85 nm. The ZnO samples with different shapes, sizes and chemical surfaces revealed some differences in biological activities. The as-synthesized nanoparticles with low toxicity exhibited desirable antioxidant and antimicrobial activities. This study showed that different parts of C. colocynthis fruit are suitable fuels to prepare ZnO-NPs and could be extended to synthesize other related metal oxide materials by microwave-assisted combustion.We have developed a simple, rapid and eco-friendly process to prepare ZnO nanocrystals using bio-fuels of fruit, seed and pulp extracts of


As a supercapacitor electrode, the nitrogen-doped porous carbons showed specific capacitance of 159 F·g−1 at 0.5 A·g−1. It also exhibited high cyclic stability with 94.8% retention of the initial specific capacitance over 1000 charge-discharge cycles at 1.0 A·g−1. The results suggest that these nitrogen-containing bio-phenol resin provide a new source of nitrogen-doped porous carbon for high-performance supercapacitor electrodes.Nitrogen hybridization is an attractive way to enhance the wettability and electric conductivity of porous carbon, which increases the capacitance of carbon-based supercapacitor, however, there is lack of low-cost methods to prepare the nitrogen-doped porous carbon materials. Herein, a novel facile nitrogen-containing bio-phenolic resin was synthesized by polymerization of the carbamate bio-oil, Phenol and paraformaldehyde. As a precursor of nitrogen-doped porous carbon, the nitrogen-containing bio-phenol resin was activated by the one-step molten-salt method. The resultant nitrogen-doped porous carbon showed a high specific surface area up to 1401 m A supercapacitor is a new generation of energy storage device, and its core component is electrode material. Among the electrode materials, porous carbon materials were the first to be studied and the most mature for technical application ,2. The iIn recent years, with the development of sustainable chemical technology, the cheap bio-oil, which is generally produced by fast pyrolysis of rich lignocellulosic biomass, can often be used as partial substitute of phenol to synthesize bio-based phenolic resin ,13,14,15In this study, we present a novel synthesis of nitrogen-containing phenol resin by amino esterification bio-oil. The nitrogen-containing phenolic resin was facilely synthesized by phenol, formaldehyde and the amino esterification bio-oil, then, the bio-phenol resin as a precursor of nitrogen-doped porous carbon was simultaneously carbonized and activated in molten salt with one-step method. The nitrogen-doped porous carbons show excellent supercapacitor performance.Bio-oil was produced from rapid pyrolysis of Poplar Sawdust at 500 °C. Urea, Phenol and Paraformaldehyde were analytical grade and purchased from Shanghai Macklin Biochemical Co., Ltd. .The 50 g of bio-oil was added into a 3-neck boiling flask. The flask was heated by an oil bath to 130 °C until melted. An appropriate amount of urea (bio-oil to mass urea ratio of 100:50) was added and stirred evenly then heated to 150 °C and held until no ammonia escaped. After the reaction finished, the products were naturally cooled to room temperature and the black bitumen solid, called carbamate bio-oil, was obtained. A specific amount of carbamate bio-oil was added into the boiling flask, heated by oil bath to 90 °C. Phenol and poly formaldehyde  was added. The mixture was held at 90 °C for 5 h. The product became a bio-phenolic resin after cooling. 2 atmosphere. The mixture was held at 900 °C for 4 h before being allowed to cool to room temperature. The product was washed with a 0.1 mL/L solution of HCL and Deionized water repeatedly until the filtrate neutralized. The product was then dried at 80 °C for 12 h to form the porous carbon.The bio-phenolic resin was cured at 100 °C for 1 h then at 180 °C for 2 h. The 3 g of heat cured bio-phenolic resin was weighed and mixed with a precise molar ratio of NaCl-KCl-KOH (NaCl:KCl:KOH = 4:4:1) salt. The mixture was placed in a tube furnace and heated from room temperature to 900 °C in a high purity NThe molecular structure of the bio-oil after amino esterification was determined by an infrared (IR) spectrometer . The thermogravimetric (TG) analysis of the phenolic resin was carried out by the Thermogravimetric Analyzer . The nitrogen content of phenolic resins was characterized by the elemental analyzer . The morphology of the specimens was determined using emission scanning electron microscopy (SEM) . The XRD patterns of specimens were investigated with a powder diffractometer . Raman spectra were recorded on a Raman spectrometer . The pore structure of the specimen was determined by Nitrogen adsorption-desorption isotherms at 77 K on an Automatic adsorption instrument .The working electrode was prepared as follow: The porous carbon material (80 wt.%), acetylene black (10 wt.%) and polytetrafluoroethylene (10 wt.%) were used to prepare a uniform paste. The paste was then coated on a nickel foam current collector.The electrochemical analysis of the specimen was carried out using a three-electrode configuration on the electrochemical workstation  in a 6 mol/L KOH electrolyte with a range of −1 to 0 V.2, P = E/Δt, where C represents the specific capacitance based on the galvanostatic charge-discharge results of supercapacitor, while U refers to the potential change within the discharge time Δt.To further evaluate the porous carbon as an electrode in symmetrical supercapacitor device, a CR2032 coin-type cell was assembled using the porous carbon as symmetrical electrodes, using a separator with the electrolyte of 6 M KOH solution. The performance of the device in terms of its energy density (E) and power density (P), which can be estimated using the following equations: E = 1/2CU−1 increased noticeably after amino esterification. The 3180–3360 cm−1 absorption peak is the symmetric and asymmetric vibration of amino N-H, showing two adjacent strong absorption peaks, while in the bio-oil there is only the absorption peak of hydroxyl in 3300–3500 cm−1. Based on the above analysis, it can be seen that carbamate was introduced, there are reactive amides, and the N element was successfully introduced.In order to testify the chemical structure change, carbamate bio-oil was characterized by IR. Molten salt one-step activation is a preparation method for activated porous carbon. The content of nitrogen in the bio-phenolic resins is important to a property of porous carbon. After testing, the nitrogen content in the bio-phenolic resins was as high as 9%. When these bio-phenolic resins were changed into activated carbon, the nitrogen content of the activated carbon still remains 5.6%. The mapping images of C, N and O elements of the activated carbon are shown in The XRD patterns of the nitrogen-doped porous carbon was shown in 2 absorption-desorption isotherms of the porous carbon. The isotherms show the characteristics of the typical type I isotherms, indicating that the pore distribution is mainly microporous, and has a lower mesoporosity [porosity ,21. The porosity , we use In order to test the cyclic stability of the specimen, 1.0 A/g current density is used to charge/discharge 1000 times. The capacitance retention curve is shown in The electrochemical impedance spectroscopy measurement is a useful method to test the conductivity of electrode materials. Measurement results can be shown by Nyquist plot. A two carbon electrode symmetrical supercapacitor was assembled to evaluate its electrochemical performance. This study provides a novel facile synthesis method of nitrogen-containing bio-phenolic resin. The nitrogen-containing bio-phenolic resin is an ideal precursor for preparation of nitrogen-doped porous carbon. Future work should focus on enlarging the capacity of nitrogen-doped mesoporous carbon. The nitrogen-doped porous carbon electrode has excellent cycling stability and could be used for a wide range of applications in supercapacitor.


Plutella xylostella were identified by small RNAs sequencing. A total of 39 miRNAs with typical characteristics of plant miRNAs were detected, of which 24 had read counts ≥ 2 in each library. Three plant-derived miRNAs with the highest read counts were validated, and all of them were predicted to target the hemocyanin domains-containing genes of P. xylostella. The luciferase assays in the Drosophila S2 cell demonstrated that miR159a and novel-7703-5p could target BJHSP1 and PPO2 respectively, possibly in an incomplete complementary pairing mode. We further found that treatment with agomir-7703-5p significantly influenced the pupal development and egg-hatching rate when reared on the artificial diet. The developments of both pupae and adults were severely affected upon their transfer to Arabidopsis thaliana, but this might be independent of the cross-kingdom regulation of the three plant-derived miRNAs on their target genes in P. xylostella, based on expression analysis. Taken together, our work reveals that the plant-derived miRNAs could break the barrier of the insect mid-gut to enter the circulatory system, and potentially regulate the development of P. xylostella. Our findings provide new insights into the co-evolution of insect herbivore and host plant, and novel direction for pest control using plant-derived miRNAs.Plant microRNAs (miRNAs) have recently been reported to be involved in the cross-kingdom regulation of specific cellular and physiological processes in animals. However, little of this phenomenon is known for the communication between host plant and insect herbivore. In this study, the plant-derived miRNAs in the hemolymph of a cruciferous specialist  O-methylation at 3′ end by the methyltransferase Hua enhancer 1 (HEN1) being involved in plants [MicroRNAs (miRNAs) are small non-coding RNAs of 19–24 nucleotides . Proteinn plants . Animal n plants .It has been reported that plant miRNAs are able to pass through the strict gastrointestinal barrier of animals when ingested, circulating in the blood, and eventually exerting biological functions by regulating gene expression in various tissues or cells ,9,10,11.Helicoverpa zea and Spodoptera frugiperda) and coleopteran (Diabrotica virgifera virgifera) insects under the controlled feeding experiments [mTOR prevents the differentiation of larvae into queens, which develop instead as workers [mTOR network involved in caste dimorphism is also conservative in the non-social insect, Drosophila melanogaster (common fruit fly) [Schizaphis graminum and Sipha flava, 13 sorghum miRNAs (eight known and five novel) and three novel barley miRNAs have been detected and are predicted to target aphid genes involved in detoxification, and starch and sucrose metabolism [In insects, plant-derived miRNAs have been found to accumulate and move systemically in lepidopteran (eriments . Accordieriments . In hone workers . The miRuit fly) . In two tabolism .However, the concept has been controversial since the first identification of the cross-kingdom regulation of plant-derived miRNAs . Other fPlutella xylostella, a worldwide destructive pest of cruciferous vegetables [P. xylostella may have long been evolving with miRNAs derived from host plants. Therefore, in this study, we explored whether plant-derived miRNAs could be detected in the circulatory system and exert functions in P. xylostella using high-throughput sequencing, molecular, cellular and biological assays.Currently, miRNAs studies related to getables ,27,28, agetables ,30,31 angetables ,33. As aP. xylostella was maintained under laboratory conditions and on a commercially-available artificial diet  for three years without exposure to any host plant. The GC strain of P. xylostella was established by transferring the newly emerged 1st instar G88 onto Col-0 type A. thaliana until 4th instar for sampling. Insects were reared at 25 ± 1 °C, an RH of 60 ± 5% and a photoperiod of 16:8 h (L:D).The insecticide-susceptible G88 strain of Arabidopsis thaliana (Col-0 ecotype) was used in this study. Seeds were surface-sterilized for 5 min with 75% ethanol and washed five times using sterile water. Seeds were suspended in sterile water and then vernalized at 4 °C in the dark for three days before germination. Seedlings were grown at 25 °C, under a 16:8 h photoperiod, on vermiculite, and were provided with a commercial nutrient solution  every day. The intensity of light was set to 2,000 lux and RH at 65 ± 5%.th instar larvae were dissected and pooled. Hemolymph samples were collected in the lysis buffer of a Quick-RNA Tissue/Insect Microprep kit  using a glass needle, and centrifuged at 13,000× g for 1 min at room temperature for RNA extraction, using the same kit according to the manufacturer’s instructions. Sequencing libraries were constructed using the NEBNext® UltraTM small RNA Sample Library Prep Kit for Illumina, and the sequencing procedure was performed by Biomarker Technologies Corporation . Clean reads were compared to the known plant microRNA (miRNA) precursors deposited in the online miRBase . Unknown plant miRNAs were inferred by miRdeep2 software using the settings of “-g -1 -l 250 -b 0” based on the information of precursors in the genome location and their structural energies [P. xylostella genome using Bowtie [To build two small RNA (sRNA) libraries of the GC strain, for each library, the hemolymph of 30 first-day 4energies . The ideg Bowtie  with a dTM Reverse Transcription System kit . The miRNA-specific reverse primer was synthesized by adding the reverse complementary sequence of the last 8 bp of mature miRNA to the 3′ terminal of the common stem-loop structure  according to the manufacturer’s instructions. The quality and concentrations of total RNA were estimated by electrophoresis and a NanoDrop 2000 spectrophotometer . The first-strand cDNA of miRNA was synthesized using the stem-loop strategy and a GoScripttructure . The remrd instar or four larvae of the 4th instar were collected and pooled. The pooled samples were frozen in liquid nitrogen immediately and stored at −80 °C until use. For mRNA profiling, total RNA was isolated using TRIzol reagent . according to the manufacturer’s instruction. The quality and concentrations of total RNAs were estimated by electrophoresis and the NanoDrop 2000 spectrophotometer . The first-strand cDNA was synthesized using a GoScriptTM Reverse Transcription System kit . The gene specific primer pairs were presented in EF1 was used as the internal control gene for normalizing the mRNA expression levels [For the RNA extraction of each of the three biological replicates, six larvae of the 3n levels .−∆∆Ct [−∆Ct [Quantitative real-time polymerase chain reaction (qRT-PCR) assays for miRNA and mRNA were conducted on the Real time polymerase chain reaction (PCR) system . For each biological replicate, the qRT-PCR tests were conducted using three technical replicates. The reaction condition was as follows: 95 °C for 3 min, 40 cycles at 95 °C for 30 s and 58 °C for 30 s. At the end of the cycling, melting curves of the resulting PCR products were obtained by gradually increasing the temperature from 55 °C to 95 °C with an increment of 0.5 °C /5 s to check the specificity of the primers. The qRT-PCR data were analyzed using methods of 2−∆∆Ct  for compCt [−∆Ct  for compP. xylostella genome and a UTR dataset for 3,050 genes obtained from DBM-DB [Drosophila melanogaster, respectively, with a setting of minimal free energy (mfe) ≤ −15 kcal/mol.Based on the CDSs of the m DBM-DB , the potm DBM-DB  and RNAhm DBM-DB  using thP. xylostella were downloaded from DBM-DB [For transcriptome sequencing, total RNA was isolated from G88 larvae at 24 h post-injection of each of three miRNA agomirs. Larvae treated with agomir-NC were used as the control. Six larvae were pooled for each replicate, and two replicates were prepared for each treatment. A total of 8 RNA samples were sequenced and analyzed at BGI Genomics Company . Genome information of m DBM-DB . Differem DBM-DB .Drosophila S2 cell line was maintained at 24 °C in Grace medium plus 10% fetal bovine serum (FBS) . The potential miRNA target sites , and ligated into the vector in the original orientation (wild-type). Mutations were introduced at the binding region of the miRNA target sites, which were used as the negative control (mutant). The S2 cells plated in 96-well plates  were co-transfected with 100 ng of each plasmid, 100 nM agomir or agomir-NC (negative control with no similarity to the miRNAs in the miRBase), and 2 µL CellfectinII reagent . The S2 cells were analyzed for luciferase activity using the Dual-Glo® Luciferase Assay System and GloMax® 20/20 Luminometer  at 48 h post-transfection. The relative luciferase activity (firefly luciferase/renilla luciferase) of the group containing agomir-NC and the wild-type pmirGLO vector was used as our control. For each transfection, the relative luciferase activity was averaged from five biological replicates.The et sites  were addrd instar larvae of the G88 strain were selected for injection (69 nL of each miRNA agomir at 100 µM per larva) using nanoliter injection 2000 . Larvae injected with agomir-NC were used as the control group. For phenotypic investigation, three biological replicates were used, and each replicate contained 20 larvae treated with miRNA agomir or agomir-NC.The agomirs (double-strand mature miRNAs) of miR-159a, miR-166a-3p and novel-7703-5p were synthesized by Sangon Biotech  . The antTotal proteins were extracted using a phenol/SDS extraction method  and quanThe immunological blots were detected using ECL Western Blot Kit  and photographed using a Fusion FX imaging system . The density of the bands was analyzed using ImageJ (version 1.52) .t test or one-way Analysis of Variance (ANOVA) followed by Tukey’s multiple comparisons was used to determine the significant difference at p-value < 0.05.Data were presented as mean ± SD and analyzed by IBM SPSS Statistics version 21 software . Student’s http://bigd.big.ac.cn/gsa.The raw sequence data of sRNA-seq and RNA-seq have been deposited in the Genome Sequence Archive (GSA)  in the BP. xylostella were well aligned to the genome of Arabidopsis thaliana , including miR159a, miR166a-3p and novel-7703-5p . All of Brassicaceae, Fabaceae, and Rosaceae.By searching mature sequences of miR159a, miR166a-3p and novel-7703-5p with that of other species in the miRBase (version 22) , we founA. thaliana. To further test the conservative nature of miR159a, miR166a-3p and novel-7703-5p in the host plant, we used stem loop PCR to clone these miRNAs in cabbage (Brassica oleracea), oilseed rape (Brassica napus), and radish (Raphanus sativus). Their mature sequences were identical in the tested host plants  in the 3 by 48 h . Therefoegulated . Annotatlostella .Px006820), basic juvenile hormone-suppressible protein 2  and phenoloxidase subunit 2  targeted by miR159a, miR166a-3p and novel-7703-5p, respectively , miR166a-3p (135) and novel-7703-5p (100), were also relatively high. We therefore hypothesized that these plant-derived miRNAs might have reached the concentration required for their absorption in P. xylostella. However, the relative levels of these miRNAs were significantly reduced in the larva-infested leaves compared with that of fresh leaves, indicating that the feeding of insect herbivores was able to influence miRNAs expression of host plants.It is assumed that the abundance of plant miRNAs detected in animal tissues should correlate with the miRNA abundance in plant tissues unless a particular miRNA is highly stable or given priority to be absorbed in animals . For exaP. xylostella. These results suggest that the normal number of miRNAs ingested from plants may not always be fully utilized and be effectively functional in herbivores [The expression levels of targets genes of the three plant-derived miRNAs in the GC strain after ingesting plant tissues were higher than that of the G88 strain in most developmental stages, although the overall performance of the GC strain was poorer than the G88 strain. This contradicted our findings that the direct injection of high doses of miRNA agomirs led to an inhibition of target genes and significantly affected the development and reproduction of rbivores . Thereforbivores ,54, may BJHSP1 and PPO2 and even affect the physiological processes of P. xylostella. It is suggested that these exogenous miRNAs are able to resist a harsh digestive tract environment, penetrate the gut cells, and be transported to target tissues or cells via the hemolymph [O-methylation on their 3′end and protected by binding with membrane proteins [Both in vivo and in vitro experiments showed that miR159a and novel-7703-5p could alter the transcriptional levels of emolymph . The higemolymph ,56,57. Temolymph . Lepidopemolymph . To safeemolymph . Along wemolymph . Plant mproteins ,13,15,63P. xylostella. These three miRNAs shared no similarity in sequences but had a similar potential to target the genes containing hemocyanin domains, which play important roles in the hemolymph of arthropods [The homologous mature sequences of miR159a, miR166a-3p and novel-7703-5p have been found to be present and conservative in diverse cruciferous plants, which may indicate that the crucifer explores these miRNAs to interact with thropods . These rthropods ,20,21, mthropods . Overall, the natural uptake and utilization of small RNA molecules in plants by herbivorous insects can become a novel avenue to understand the complex plant-insect interactions and develop new technologies for pest management based on miRNA.


Miichthys miiuy) with yields of 0.64 ± 0.07% and 3.87 ± 0.15% of dry weight basis, respectively. ASC-MC and PSC-MC had glycine as the major amino acid with the contents of 341.8 ± 4.2 and 344.5 ± 3.2 residues/1000 residues, respectively. ASC-MC and PSC-MC had lower denaturation temperatures  compared to mammalian collagen due to their low imino acid content . ASC-MC and PSC-MC were mainly composed of type I collagen on the literatures and results of amino acid composition, SDS-PAGE pattern, ultraviolet (UV) and Fourier-transform infrared spectroscopy (FTIR) spectra. The maximum solubility of ASC-MC and PSC-MC was appeared at pH 1–3 and a sharp decrease in solubility was observed when the NaCl concentration was above 2%. Zeta potential studies indicated that ASC-MC and PSC-MC exhibited a net zero charge at pH 6.66 and 6.81, respectively. Furthermore, the scavenging capabilities on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydroxyl radical, superoxide anion radical and 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical of ASC-MC and PSC-MC were positively correlated with their tested concentration ranged from 0 to 5 mg/mL and PSC-MC showed significantly higher activity than that of ASC-MC at most tested concentrations (p < 0.05). In addition, the scavenging capability of PSC-MC on hydroxyl radical and superoxide anion radical was higher than those of DPPH radical and ABTS radical, which suggested that ASC-SC and PSC-SC might be served as hydroxyl radical and superoxide anion radical scavenger in cosmeceutical products for protecting skins from photoaging and ultraviolet damage.In this report, acid-soluble collagen (ASC-MC) and pepsin-soluble collagen (PSC-MC) were extracted from the scales of miiuy croaker ( Collagen is the most abundant protein constituting nearly 30% of all proteins in the animal body and is a primary component of the extracellular matrix . CollageAn imbalance in pro-oxidant/antioxidant can cause oxidative stress, which further trigger the accumulated reactive oxygen species (ROS) production and result in cell damage and many health disorders, such as skin damage, diabetes mellitus, cancer and inflammatory diseases ,11. TherMiichthys miiuy) is an important and highly consumed aquaculture species in China and Japan and it has been widely cultured since late 1990s because of its fast growth, various feeding habit and high medicinal and economic values [M. miiuy) were prepared and their physicochemical and antioxidant properties were characterized for their potential applications in the cosmetic and biomedical industries.Fish scales are composed of protein and collagen of connective tissue (41 to 81%) and calcium-deficient hydroxyapatite. For now, approximately 49,000 tons of fish scales are generated in the de-scaling process of aquatic products. However, large quantities of scales are discarded as waste during processing and filleting due to lower economic value, which give rise to some additional ecological environmental problems especially in developing countries. Effective use of those resources not only solves the problem of environmental pollution but also increases economic returns for the fishery industry. Therefore, collagens have been isolated from scales of some kinds of fish ,18,19,20c values . TherefoChemical compositions of scale from miiuy croaker, as well as the ASC-MC and PSC-MC derived from them are presented in M. miiuy) with yields of 0.64 ± 0.07% and 3.87 ± 0.15% of dry weight basis, respectively. The yield of PSC-MC was 6.05-fold higher than that of ASC and it could be supposed that there were many interchain cross-links at the telopeptide region, leading to the low solubility of collagen in acid [ASC-MC and PSC-MC were isolated from the scales of miiuy croaker , Catla catla (353 residues/1000 residues) [L. rohita (361 residues/1000 residues), C. catla (353 residues/1000 residues) [The amino acid compositions of type I collagen from calf skin (CSC), ASC-MC and PSC-MC from the scales of miiuy croaker were expressed as amino acid residues per 1000 total amino acid residues and presented in esidues) , redlip esidues) . The Glyesidues) , Nile tiesidues) , redspotesidues)  and snakesidues)  but loweesidues) , L. rohiesidues) , bigheadesidues)  and crocesidues) . C. catla [As shown in C. catla  but signC. catla . The imiC. catla , snakeheC. catla , redspotC. catla  and blacC. catla  but signC. catla .Pyrrolidine rings of imino acid enforced constraints on the conformation of the polypeptide chain and helped to strengthen the thermal stability of triple helix. It has been verified that Hyp has played an important role in stabilizing the triple-stranded helix of collagen by hydrogen bonds ,25. Ther2α2). This finding was agreement with the scale collagens from tilapia [L. rohita and C. catla [SDS-PAGE pattern is commonly applied to determine the type and composition of collagen on the subunit composition, electrophoretic mobility and intensity of the band. Similar protein patterns of ASC-MC, PSC-MC and type I collagen from calf skin (CSC) were observed in  tilapia , snakehe tilapia , Japanes tilapia , L. rohiC. catla .M. miiuy) are presented in Peptide hydrolysis patterns of ASC-MC and PSC-MC from the scales of miiuy croaker (2 in polypeptides chains of collagens [It is well known that the maximum absorption wavelength of protein in the near ultraviolet region is 280 nm because of the absorbance (280 nm) of aromatic amino acids such as Phe, Trp and Tyr ,22. The ollagens , body waollagens  and chanollagens .FTIR spectrum is a powerful technique to research the structure and of collagens and configuration of polypeptide chain and the frequencies relate to the nature of the molecular bonds and their structure and chemical environment . Collage−1 and the wavenumber would move to lower frequency if the N-H group participated in the formation of a hydrogen-bond [−1. The data illustrated that some N-H groups in ASC-MC and PSC-MC contributed to the formation of hydrogen bonding and the hydrogen-bonding numbers of ASC-MC were more than that of PSC-MC. However, the amide A wavenumbers of ASC-MC and PSC-MC were lower than that of CSC (3426 cm−1), which indicated that the structure stability of ASC-MC and PSC-MC was weaker than that of CSC. The amide B band is related to asymmetric stretch vibrations of −1, respectively. The result indicated that the free The band of amide A is bound up with the N-H stretching frequency. The wavenumber of a free N-H stretching vibration is located next to the range 3400–3440 cmgen-bond ,38. Figuterminal . The wav−1 is primarily associated with the C=O stretching vibration along the polypeptide backbone or a hydrogen bond coupled with COO– and the decrease of molecular order will make the peak shift to lower wavenumber [−1 and slight lower wavenumber (1655 cm−1) was found for PSC-MC. The result indicated that partial telopeptides were degraded by pepsin during the preparation process of PSC-MC, which caused the missing of active amino acids  at telopeptide region of PSC-MC molecular [Amide I, amide II and amide III bands are bound up with the triple helical structure of collagen, resulting from C=O stretching, N–H bending and C–H stretching, respectively ,26. The venumber . Amide Iolecular . −1, which specifies the number of NH groups involved in hydrogen bonding with the adjacent α-chain; therefore, the lower wavenumber of the amide II band is related to the increased of hydrogen bonds by NH groups, which is attributed to collagen’s higher structure order [−1, respectively, which indicated that the hydrogen bonding in CSC and ASC-MC was more than that of PSC-MC and the finding is consistent with the result of peptide hydrolysis patterns.The amide II band representing the N–H bending vibration coupled with C–N stretching vibration generally occurs in the range of 1550–1600 cmre order . The wav2 groups from the Gly backbone and Pro side-chains, which is weak and associated with the triple helix structure of collagen [−1, respectively. The result indicated that hydrogen bonds were involved in CSC, ASC-MC and PSC-MC. In addition, the intensity ratio between Amide III band and 1450 cm−1 band has been used to elucidate the triplehelical structure of collagen and the absorption ratio between amide III  and 1452 cm−1 (CSC), 1451 cm−1 (ASC-MC) or 1448 cm−1 (PSC-MC) bands was approximately equal to 1.0, which confirmed that ASC-MC and PSC-MC have maintained a high extent of intact triple helix structures. In addition, the amide I and amide A bands on ASC-MC and PSC-MC suggested that the structure of ASC-MC was more stable than that of PSC-MC due to more hydrogen-bonding and partial telopeptides in ASC-MC molecular but the structures of ASC-MC and PSC-MC were more unstable than that of CSC on the information of their FTIR spectra.Amide III band absorption was arisen from wagging vibrations of CHcollagen . Figure Collagen consists of amino acids wound together to form triple-helices to form of elongated fibrils and the triple helix structure could be depolymerized and transformed to the unordered coil configuration if the intramolecular hydrogen bond was broken by high temperature, which is along with the changes of physical characteristics, such as solubility decrease, precipitation and viscosity reducing. Therefore, viscosity measurement is often applied to research the thermos stability of collagen ,8.Td) is the temperature at which the triple-helix structure of collagen deforms to a random coil structure. The Td values of ASC-MC and PSC-MC were 32.2 and 29.0 °C, which were similar to those of some warm and tropical fish species, such as skipjack tuna (29.7 °C), paper nautilus (27 °C), ocellate puffer (28 °C), eel (29.3 °C), Japanese seabass (26.5 °C) and ayu (29.7 °C) [Td values of ASC-MC and PSC-MC were lower than those of CSC (35.9 °C). The finding further confirmed that the helix structures of ASC-MC and PSC-MC were more unstable than those of collagens from mammals. A low Td value is an undesirable property in the manufacturing process and for biomaterials because denaturation drastically changes the biological, mechanical and physicochemical properties of collagen [Td and viscosity of PSC-MC were slightly lower than this of ASC-MC, which might be caused by MW reduction in the telopeptide region induced by pepsin hydrolysis.As shown in 29.7 °C)  and high29.7 °C) . Howevercollagen . At prescollagen . The Td Solubility of collagen is the most important factor and excellent index for their functionality. Knowledge of collagen solubility can give useful information on the potential utilization of proteins and their functionality, especially in foams, emulsions and gels . In addiI) because Collagen carries a positive or negative charge [I, attractive forces predominate and molecules tend to associate, resulting in insolubility. C. catla [Is of ASC-MC and PSC-MC were about pH 7 and the data were in agreement with previous reports that the collagen pIs were usually at pH 6–9 [Collagen to be soluble should be able to interact as much as possible with the solvent. Collagen-water interactions increase at pH values higher or lower than the isoelectric point  [I values might due to amino acid sequences and distribution of amino acid residues.Zeta potential, also known as electrokinetic potential, is the potential difference across phase boundaries between solids and liquids and often used to describe double-layer properties of a colloidal dispersion . Therefoctively) , ASC andctively)  and PSC ctively) . The difw/v)) suggested that there were some differences in the primary structures between ASC-MC and PSC-MC. Previous reports suggested that collagens with interconnectivity, fibrillary and sheet-like film structures have the potentiality to be used in new tissue formation, cell seeding, growth, wound healing and mass transport and migration [Ultrastructure and surface area of collagen are important to evaluate its potential applications in biomedicine and biomedicine engineering ,30. ScanOxidative stress is associated with the pathogenesis of many chronic diseases and excessive free radicals generated in metabolism are a potential reason for the oxidative stress of human body ,47. Reacp < 0.05) and the hydroxyl radical scavenging activity of PSC-MC showed the same trend and was significantly higher than those of ASC-MC and CSC at the same concentrations except the concentrations of 0.5 mg/mL (p < 0.05). Furthermore, PSC-MC showed significantly higher superoxide anion radical and ABTS radical scavenging activity than PSC-MC did at the same concentrations (p < 0.05). However, there were no significant difference between ASC-MC and CSC on hydroxyl radical, superoxide anion radical and ABTS radical scavenging activities at most tested concentrations (p > 0.05). In addition, the radical scavenging activities of ASC-MC, PSC-MC and CSC were significantly lower than those of the positive control of ascorbic acid and glutathione (GSH), which was agreed with the previous reports that small molecular including oligopeptides (2–9 amino acid residues) showed high radical scavenging activities than macromolecules because they were easily accessible to active radicals to provide potential effects in reaction mixture [The antioxidant properties of ASC-MC and PSC-MC were evaluated using DPPH radical, hydroxyl radical, superoxide anion radical and ABTS radical scavenging assays and shown in  mixture ,52. Moreover, the scavenging capability of PSC-MC on hydroxyl radical B and supApostichopus japonicus showed protective effects against ultraviolet radiation-induced skin photoaging [The skin aging process can be divided into intrinsic aging and photoaging. Skin photoaging is a premature skin-aging damage after repeated exposure to ultraviolet (UV) radiation, mainly characterized by oxidative stress and inflammatory disequilibrium, which makes skin show the typical symptoms of photoaging such as coarse wrinkling, dryness, irregular pigmentation and laxity . ROS areotoaging . Hou et otoaging . Sun et otoaging . TherefoM. miiuy) were obtained from Zhejiang Hailisheng Group Co. Ltd., in Zhoushan City, Zhejiang Province of China. High molecular weight markers and type I collagen from calf skin (CSC) were used as the standards and bought from Sigma-Aldrich . All other reagents used were of analytical grade.The scales of miiuy croaker (w/v) and stirred for 6 h and NaOH solution was replaced each 3 h. Afterwards, the scales were rinsed using cold tap water until the pH value of washing water got to 7.0–7.5 and demineralized using EDTA-2Na  with a material/liquid ratio of 1:10 (w/v) and stirred for 48 h and EDTA-2Na solution was renewed every 12 h. The pretreated scales were cleaned with a scale/cold tap water ratio of 1:20 (w/v) for three times.The extraction procedure of scale collagens of miiuy croaker was according to the method described by Matmaroh et al. . The scaw/v) for 48 h. The extracting solution was filtered using a cheesecloth and the collagen was precipitated from the filtrate using 2.5 M NaCl solution. The precipitates were collected by centrifugation at 20,000× g for 30 min at 4 °C, re-dissolved in a minimum volume of 0.5 M acetic acid solution and dialyzed against 25 volumes of 0.1 M acetic acid solution for 12 h. Thereafter, the dialyzed solution was dialyzed against 25 volumes of distilled water for 48 h and distilled water was changed each 12 h. The final dialysate was lyophilized.The pretreated scales were soaked in 0.5 M acetic acid solution with a material/liquid ratio of 1:15 (w/w) at a solvent/scale ratio of 15:1 (v/w) for 48 h at 4 °C. Thereafter, other processes were carried with the identical manner as the ASC-MC preparation.The scale residues from ASC-MC preparation were soaked in 0.5 M acetic acid solution containing 1% porcine pepsin  method (2003) with the method numbers of 950.46B, 920.153 and 960.39 (a), respectively. Protein content was measured using the Kjeldahl method and an auto protein analyzer . The converting factor of 6.25 was used for calculation of protein content .Tested samples were hydrolyzed in 6 M HCl at 110 °C for 24 h and the hydrolysates were vaporized and the residues were dissolved in 25 mL citric acid buffer solution. An aliquot of 0.05 mL was applied to an automated amino acid analyzer . Then the degrees of Pro and Lys hydroxylation (%) were calculated as follows:Degrees of Pro hydroxylation (%) = Hyp content/(Hyp content + Pro content) × 100%.Degrees of Lys hydroxylation (%) = Hyl content/(Hyl content + Lys content) × 100%.w/v) SDS prior to incubation at 85 °C for 1 h. The mixture was centrifuged at 5,000× g for 10 min for removing undissolved debris. The samples (about 20 μL) were mixed with the sample loading buffer  at the ratio of 4:1 (v/v) in the presence of β-ME, then applied to sample wells and electrophoresed in an electrophoresis instrument . The electrophoresis was carried out for about 4 h at a constant voltage of 100 V. After electrophoresis, gel was stained with 0.1% (w/v) Coomassie blue R-250 in 45% (v/v) methanol and 10% (v/v) acetic acid.Electrophoretic patterns of ASC-MC and PSC-MC were determined using the previous method , using 7w/w) and the hydrolysis was terminated in boiled water for 5 min after the addition of SDS-PAGE sample buffer. SDS-PAGE with 12.0% separating gels was used to measure the molecular weight of peptides.Peptide hydrolysis patterns of ASC-MC and PSC-MC were measured on the method described by Wu et al. . Collagew/v).The UV adsorption spectra of ASC-MC and PSC-MC were recorded using the method of Yu et al. , using aw/w) was pressed into a disk for spectrum recording. The IR spectra in the range of 4000–400 cm−1 with automatic signal gain were collected in 32 scans at a resolution of 4 cm−1 and were ratioed against a background spectrum recorded from the clean empty cell.The IR spectra of ASC-MC and PSC-MC were recorded in KBr disks with a FTIR spectrophotometer . The mixture at a sample to potassium bromide (KBr) ratio of 1:100 (w/v) and 500 mL solutions were subjected to viscosity measurement using a NDJ-8S viscometer  with appropriate spindles (from No.4 to No.1) and an appropriate speed. All the sample solutions were heated from 4 to 44 °C with a heating rate of 4 °C/min and the solution was held for 30 min prior to viscosity determination at the designated temperature. The relative viscosity was calculated in comparison with that obtained at 4 °C and Td was defined as the temperature at which relative viscosity was 0.5.Viscosity of ASC-MC and PSC-MC was measured using the previous method . All theg for 15 min at 4 °C and the resulting supernatants were used for measuring solubility of collagen.Effects of pH and NaCl concentration on the collagen solubility were measured using the previous method . Collageg for 60 min at 4 °C. For all the samples, protein content in the supernatant was measured. Then the relative solubility was calculated in the comparison with that of be obtained at the pH giving the highest solubility. Sample solution (8 mL) was transferred to a 50 mL centrifuge tube and the pH was adjusted with either 6 M NaOH or 6 M HCl to obtain the final pH ranging from 1 to 11. The volume of solution was made up to 10 ml by deionized water previously adjusted to the same pH as the sample solution. The solution was centrifuged at 15,000× g for 60 min at 4 °C.Sample solution (5 mL) was mixed with 5 mL of NaCl in 0.5 M acetic acid at various concentrations to give the final concentrations of 0%, 1%, 2%, 3%, 4%, 5% and 6%. The mixture was stirred continuously at 4 °C for 30 min, followed by centrifuging at 15,000× Is of ASC-MC and PSC-MC were determined from the pH value that resulted in a zero zeta potential.Zeta potentials of ASC-MC and PSC-MC were measured on the previous method . ASC-MC w/v), followed by dialyzing against distilled water. The collagen was lyophilized in a freeze dryer  and the sample was sputter coated for 90 s with gold using a JEOL JFC-1200  fine coater. The morphologies of the electro spun fibers and membrane were observed using Hitachi TM-1000 .The morphological characteristics of ASC-MC and PSC-MC were studied by SEM using Hitachi TM-1000 . Collagen was re-dissolved in 0.5 M acetic acid at a concentration of 5%  scavenging activity and lipid peroxidation inhibition assays were performed according to previously reported methods ,59.p < 0.05) was used to measure the significant differences between the parameters means.All experiments were carried out in triplicate. An ANOVA test using the software of SPSS 19.0  as applied to compare the average values of each treatment. Duncan’s multiple range test (M. miiuy) were isolated and characterized. Amino acid composition, SDS-PAGE pattern, UV spectra and FTIR confirmed that ASC-MC and PSC-MC were mainly composed of type I collagen. The antioxidant capacities of ASC-MC and PSC-MC were positively correlated with their tested concentration ranged from 0 to 5.0 mg/mL and the radical scavenging activity of PSC-MC was significantly higher than that of ASC-MC at most tested concentrations (p < 0.05). The present result suggested that ASC-SC and PSC-SC from the scales of miiuy croaker could be served as substitutes of skin collagens from mammalian and aquatic products in cosmeceutical products for protecting skins from photoaging and ultraviolet damage by scavenging reactive oxide species. Therefore, this study provides scientific basis for the medical application of scale collagens of miiuy croaker (M. miiuy).In the experiment, acid-soluble collagen (ASC-MC) and pepsin-soluble collagen (PSC-MC) from the scales of miiuy croaker (